The central focus of the renewal will be on understanding how CFTR interacts with other proteins. The concept of how transport proteins work together to coordinate the transport of solutes and water across epithelial cell membranes has been a question fundamental to epithelial physiology. A combination of molecular and physiological experiments will give us the opportunity to address long standing questions regarding transporter homeostasis To address this issue we will focus on proteins that bind to CFTR and enable it to function in a macro-molecular complex. During the previous budget period we have discovered a new protein called CAL (CFFR associated ligand). CAL is a new member of the PDZ domain protein family. in addition to a single PDZ domain, CAL is predicted to have other protein interacting regions (coiled-coil domains). CAL binding to the cytoplasmic carboxy-terminus of CFTR via its PDZ domain organizes CFTR into protein clusters. Apical localization indicates that CAL serves as a scaffold protein that interacts with CFTR at the epithelial plasma membrane. CFTR, an epithelial Cl- channel defective in cystic fibrosis (CF), has been functionally linked to other proteins. The Project has three goals to determine the functional consequences of the interaction between CAL and CFTR, to identify the structural domains of CFTR critical for the regulatory interaction between CFTR and CAL and to determine what are the functional consequences of multiple PDZ domain proteins that bind to CFTR. These studies will employ a combination of approaches. The ultimate goal of these studies is to provide novel insights into how CFTR functions in a macromolecular complex. The reasons for gaining more information on the terminal amino acids of CFTR and proteins such as CAL that bind to it are at least two-fold. First, CFTR is a transport protein that functions in a variety of epithelial cells. More importantly, information how CFTR interacts with other proteins will increase our information on how mutant CFTR causes CF. This is particularly important to our understanding of lung pathobiology whose severity of disease often does not correlate with genotype. Finally, the novel proteins that bind to CFTR provide important targets for pharmacologic therapies.